1. Field of the Invention
The present invention relates to methods for sterilizing preparations of glycosidases to reduce the level therein of one or more active biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and/or single or multicellular parasites. The present invention particularly relates to methods of sterilizing preparations of glycosidases, such as alpha-glucosidase or alpha-galactosidase, with irradiation.
2. Background of the Related Art
The principal foods upon which an organism, such as a human, survives can be broadly categorized as carbohydrates, fats and proteins. These substances, however, are useless as nutrients without the process of digestion to break down foods into chemical components that are sufficiently small to be absorbable in the digestive tract.
Digestion of carbohydrates begins in the mouth and stomach. Saliva contains the enzyme ptyalin (an alpha-amylase), which hydrolyses starch into maltose and other small polymers of glucose. The pancreatic alpha-amylase is similar to the salivary ptyalin, but several times as powerful. Therefore, soon after chyme empties into the duodenum and mixes with pancreatic juice, virtually all of the starches are converted into disaccharides and small glucose polymers. These disaccharides and small glucose polymers are hydrolysed into monosaccharides by intestinal epithelial enzymes, such as intestinal sucrase, intestinal maltase, and intestinal lactase.
Digestion of proteins begins in the stomach. The ability of pepsin to digest collagen is especially important because collagen is a major constituent of the intercellular connective tissue of meats. For other glycosidases to penetrate meats and digest various cellular proteins, the collagen fibers must first be partially digested by pepsin. People who lack peptic activity in the stomach will experience poor absorption of ingested meats because there is poor penetration by these other glycosidases.
Most protein digestion results from the actions of the pancreatic proteolytic enzymes. Proteins leave the stomach in the form of proteoses, peptones and large polypeptides, and are digested into dipeptides, tripeptides and the like by pancreatic proteolytic enzymes or polypeptidases. Trypsin and chymotrypsin split protein molecules into smaller polypeptides at specific peptide linkages, whereas carboxypolypeptidase cleaves amino acids from the carboxyl ends of polypeptides. The zymogen proelastase is converted to the active protease elastase, which in turn digests elastin fibers that hold together most meat.
Further digestion of polypeptides takes place in the intestinal lumen. Aminopolypeptidase and several other polypeptidases split large polypeptides into dipeptides, tripeptides and amino acids, which are then transported into enterocytes that line the intestinal villi. Inside the enterocytes, other polypeptidases split any remaining peptides into their constituent amino acids, which then enter the blood.
Digestion of fats first requires emulsification by bile acids and lecithin, which increase the surface area of the fats up to 1000-fold. Because lipases are water-soluble glycosidases that can bind only on the surface of a fat globule, this emulsification process is important for the complete digestion of fat. The most important glycosidase in the digestion of triglycerides is pancreatic lipase, which breaks these down into free fatty acids and 2-monoglycerides. After these free fatty acids and monoglycerides enter the enterocytes, they are generally recombined into new triglyerides. A few monoglycerides, however, are further digested by intracellular lipases into free fatty acids.
Glycosidases are required to digest or break down saccharide units (usually polysaccharides). For example, glycosidases are required to digest or breakdown the saccharide untis that are covalently attached to proteins so that proteases can then gain access to the protein for cleavage of the individual amino acids therefrom, to thereby promote absorption of the resulting amino acids.
A number of glycosidases are present in the human gastrointestinal tract, but, to date, only some have been isolated and sufficiently characterized. Many glycosidases, however, have been isolated and characterized from plant and microbial sources, which, to some extent, has provided a roadmap for studies of animal glycosidases. Thus, glycosidases are known that cleave and remove O-linked sugar units from glycoproteins, and from glycolipids and polysaccharides, for example, alpha-N-acelylgalactosaminidase. Other glycosidases include N-acetylneuraminic acid aldolase, beta(1-4) galactosidase, beta(1-3,6) galactosidase, beta(1-3,4,6) galactosidase, beta(1-6)galactosidase, alpha(1-3,6) galactosidase, beta-glucosaminidase, alpha-mannosidase, alpha(1-3,4) fucosidase, alpha(1-2,3,4) fucosidase, alpha(1-2) fucosidase, beta(1-2) xylosidase, beta(1-4) xylosidase, peptide-N4-(acetyl-beta-glucosaminyl)-asparagine amidase (EC 3.5.1.52) hexosaminidase, beta-N-acetylhexosaminidase, alpha(2-3,6,8,9) neuraminidase, various sialidases, such as N-acetylneuraminate glycohydrolase (EC 3.2.1.18), various glycoamidases, alpha-mannosidase, and beta-mannosidase.
The nomenclature of glycosidases typically indicates the type of linkages that are cleaved by the enzyme. For example, beta(1-3,6) galactosidase cleaves only beta(1-3,6) galactose linkages, but not beta(1-4) galactose linkages.
Reaction conditions and substrate specificities vary greatly, depending on the particular glycosidase. For example, a given glycosidase may be ineffective when sialic acid is present on N-linked oligosaccharides. The optimal pH activities of glycosidases also vary according to the particular glycosidase, with some being optimally active at acidic pH values, others at values near neutral pH, and yet others at alkaline pH values.
Preparations of glycosidases may be required for administration to humans and other animals when, for example, there is a genetically caused disease, such as lack of endogenous glycosidases, or lack of active glycosidases. One genetic disease characterized by a partially or completely inactive glycosidase is Fabry disease, which afflicts about one in 40,000 people in the United States. Fabry disease is an X-linked lysosomal disorder in which the patient""s body does not have a normal ability to break down a fatty substance, globotriaosylceramide (also known as Gb3 or ceramidetrihexoside). Gb3 is present in membranes of many cell types, including the membranes of red blood cells. Roughly 1% of one""s red blood cells are replaced each day, which means that a significant amount of Gb3 requires degradation each day.
One of the major lysosomal enzymes involved in the degradation of Gb3 is alpha-galactosidase A (xe2x80x9cxcex1-gal Axe2x80x9d), which is either partially or completely inactive in patients with Fabry disease. As a result, Gb3 accumulates in lysosomes throughout the patient""s body, which impairs (clogs blood vessels with built-up Gb3) organs and body parts that depend on proper functioning of small blood vessels, such as kidneys, heart, nervous system, and skin.
The most common symptom of Fabry disease is pain, which may occur in the form of periods of intense burning, or sharp, shooting pain. The pain may be brought on by such events as exercise, fever, fatigue, stress, and/or exposure to temperature changes. In addition, many patients with Fabry disease are unable to perspire, which causes further discomfort with exercise or exposure to high temperatures. Periods of pain are most common in childhood, but also may not present until the 20s, when sufficient Gb3 has accumulated. In some patients, the pain subsides with increasing age, and in others the pain increases.
Another common symptom of Fabry disease is a spotted, dark-red skin rash that most commonly occurs from the belly button down to about the knees (the xe2x80x9cbathing trunkxe2x80x9d rash). Other symptoms that can be associated with Fabry disease, and that can exhibit extremes in variability from patient to patient, include chronic bronchitis and shortness of breath, swelling of the legs, diarrhea, osteoporosis, growth retardation, delayed puberty, and development of a hazy or opaque cornea.
Once the buildup of Gb3 reaches a critical level, symptoms begin to appear more regularly, typically including problems with function of the heart, the circulatory system, and the kidneys. Thus, common problems include heart and circulatory malfunctions, such as high blood pressure, heart attack, heart failure, mitral valve prolapse, cardiac arrhythmia, stroke, and kidney malfunctions, such as renal failure requiring dialysis of the patient.
Recently, treatment for Fabry disease has included infusion of a preparation of xcex1-galactosidase A (Replagal) to patients, with reported stabilization or improvement in renal function, decreased pain, and reduction in mass of the heart (Schiffmann, et al., xe2x80x9cEnzyme Replacement Therapy in Fabry Disease: A Randomized Controlled Trial.xe2x80x9d JAMA, Jun. 6, 2001, Vol.285, No. 21, pp. 2743xe2x80x942749.).
Another genetic disease characterized by inactive glycosidases is Glycogen Storage Disease Type II (also called GSD II, acid maltase deficiency, AMD, Pompe disease, and alpha-glucosidase deficiency), in which an autosomal recessive mutation is expressed for the lysosomal enzyme acid alpha-glucosidase (acid maltase). Although prognosis and specific symptomology vary according to the subtype (infantile form [type a], childhood form [type b], or adult form [type c]), some of the general effects are roughly the same across the various types, in particular, respiratory and cardiac failure due to massive accumulations of glycogen in the respiratory muscles and in the heart itself. Other possible symptoms include muscle weakness and degeneration, and enlargement of the heart, liver and tongue. Normally, acid alpha-glucosidase breaks down excess glycogen in a cell; however, this function is blocked in cells of the GSD II patient to produce abnormal, excess accumulation of glycogen, which causes the muscle failure in respiratory muscles and in heart muscle. At least one report (H. Van den Hout, et al., 2000. xe2x80x9cRecombinant human alpha-glucosidase from rabbit milk in Pompe patients.xe2x80x9d Lancet 356: 397-398 Jul. 29, 2000 issue)) has shown successful treatment of infantile GSD II patients with administration of recombinant human acid alpha-glucosidase.
Another medical use for glycosidases commonly occurs in response to gas (flatulence) that is caused by the human digestive tract""s inability to degrade small disaccharides, trisaccharides, etc. These small oligosaccharides are then fermented by intestinal flora to produce undesired, excess gas. Such offending oligosaccharides are prevalent in cruciferous vegetables, such as broccoli, cabbage, cauliflower, and brussels sprouts, as well as in beans and lentils. Removal of the offending oligosaccharides in beans and lentils may be effected by bringing them to a boil to break the outer husks, adding a small quantity of baking soda (sodium bicarbonate) and turning off the heat, allowing the xe2x80x9cbrewxe2x80x9d to sit for 4-8 hours, and thoroughly rinsing the resulting mixture several times with fresh tap water prior to completion of the cooking process. A simpler solution, however, involves administration of products containing alpha-galactosidase, such as Beano(trademark), concurrently with consumption of a gas-producing food. These products decrease gas production by breaking down the oligosaccharides prior to their reaching intestinal fermenting sites, but do not degrade fiber molecules.
Preparations of glycosidases that are prepared for human, veterinary, diagnostic and/or experimental use may, however, contain unwanted and potentially dangerous biologically active contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multicellular parasites, prions or similar agents responsible, alone or in combination, for TSEs. Consequently, it is of utmost importance that any biologically active contaminant or pathogen in the preparation be inactivated before the product is used. This is especially critical when the glycosidase preparation is to be administered directly to a patient, for example in human therapy. This is also critical for the various enzyme preparations that are prepared in media which contain various types of plasma and/or plasma derivatives or other biologic materials and which may contain prions, bacteria, viruses and other biological contaminants or pathogens.
Most procedures for producing preparations of enzymes have involved methods that screen or test the preparation for one or more particular biological contaminants or pathogens rather than removal or inactivation of the contaminant(s) and/or pathogen(s) from the preparation. Preparations that test positive for a biological contaminant or pathogen are merely not used. Examples of screening procedures include the testing for a particular virus in human blood from blood donors. Such procedures, however, are not always reliable and are not able to detect the presence of certain viruses, particularly in very low numbers, and in the case of as yet unknown viruses or other contaminants or pathogens that may be in blood. This reduces the value or certainty of the test in view of the consequences associated with a false negative result. False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the preparation is contaminated. Moreover, to date, there is no reliable test or assay for identifying prions within a biological material that is suitable for screening out potential donors or infected material. This serves to heighten the need for an effective means of destroying prions within a biological material, while still retaining the desired activity of that material. Therefore, it would be desirable to apply techniques that would kill or inactivate biological contaminants and pathogens during and/or after manufacturing the preparation of glycosdisases.
The importance of these techniques is apparent regardless of the source of the biological material. All living cells and multi-cellular organisms can be infected with viruses and other pathogens. Thus the products of unicellular natural or recombinant organisms or tissues carry a risk of pathogen contamination. In addition to the risk that the producing cells or cell cultures may be infected, the processing of these and other biological materials creates opportunities for environmental contamination. The risks of infection are more apparent for multicellular natural and recombinant organisms, such as transgenic animals. Interestingly, even products from species as different from humans as transgenic plants carry risks, both due to processing contamination as described above, and from environmental contamination in the growing facilities, which may be contaminated by pathogens from the environment or infected organisms that co-inhabit the facility along with the desired plants. For example, a crop of transgenic corn grown out of doors, could be expected to be exposed to rodents such as mice during the growing season. Mice can harbour serious human pathogens such as the frequently fatal Hanta virus. Since these animals would be undetectable in the growing crop, viruses shed by the animals could be carried into the transgenic material at harvest. Indeed, such rodents are notoriously difficult to control, and may gain access to a crop during sowing, growth, harvest or storage. Likewise, contamination from overflying or perching birds has to potential to transmit such serious pathogens as the causative agent for psittacosis. Thus any biological material, regardless of its source, may harbour serious pathogens that must be removed or inactivated prior to the administration of the material to a reicipient.
In conducting experiments to determine the ability of technologies to inactivate viruses, the actual viruses of concern are seldom utilized. This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal. In their place, model viruses of the same family and class are used. In general, it is acknowledged that the most difficult viruses to inactivate are those with an outer shell made up of proteins, and that among these, the most difficult to inactivate are those of the smallest size. This has been shown to be true for gamma irradiation and most other forms of radiation as these viruses"" diminutive size is associated with a small genome. The magnitude of direct effects of radiation upon a molecule are directly proportional to the size of the molecule, that is the larger the target molecule, the greater the effect. As a corollary, it has been shown for gamma-irradiation that the smaller the viral genome, the higher the radiation dose required to inactive it.
Among the viruses that may contaminate both human and animal-derived preparations, the smallest, and thus most difficult to inactivate, belong to the family of Parvoviruses and the slightly larger protein-coated Hepatitis virus. In humans, the Parvovirus B19, and Hepatitis A are the agents of concern. In porcine-derived materials, the smallest corresponding virus is Porcine Parvovirus. Since this virus is harmless to humans, it is frequently chosen as a model virus for the human B19 Parvovirus. The demonstration of inactivation of this model parvovirus is considered adequate proof that the method employed will kill human B19 virus and Hepatitis A, and by extension, that it will also kill the larger and less hardy viruses such as HIV, CMV, Hepatitis B and C and others.
More recent efforts have focussed on methods to remove or inactivate contaminants in the products. Such methods include heat treating, filtration and the addition of chemical inactivants or sensitizers to the product.
Heat treatment requires that the product be heated to approximately 60xc2x0 C. for about 70 hours which can be damaging to sensitive products. In some instances, heat inactivation can actually destroy 50% or more of the biological activity of the product.
Filtration involves filtering the product in order to physically remove contaminants. Unfortunately, this method may also remove products that have a high molecular weight. Further, in certain cases, small viruses and similarly sized contaminants and pathogens, such as prions, may not be removed by the filter.
The procedure of chemical sensitization involves the addition of noxious agents which bind to the DNA/RNA of the virus and which are activated either by UV or other radiation. This radiation produces reactive intermediates and/or free radicals which bind to the DNA/RNA of the virus, break the chemical bonds in the backbone of the DNA/RNA, and/or cross-link or complex it in such a way that the virus can no longer replicate. This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and cannot be administered to a patient.
Irradiating a product with gamma radiation is another method of sterilizing a product. Gamma radiation is effective in destroying viruses and bacteria when given in high total doses (Keathly et al., xe2x80x9cIs There Life After Irradiation? Part 2,xe2x80x9d BioPharm July-August, 1993, and Leitman, Use of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease,xe2x80x9d Transfusion Science 10:219-239 (1989)). The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, enzymes, protein and protein-containing products. In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman). U.S. Pat. No. 4,620,908 discloses that protein products must be frozen prior to irradiation in order to maintain the viability of the protein product. This patent concludes that xe2x80x9c[i]f the gamma irradiation were applied while the protein material was at, for example, ambient temperature, the material would be also completely destroyed, that is the activity of the material would be rendered so low as to be virtually ineffectivexe2x80x9d. Unfortunately, many sensitive biological materials, such as monoclonal antibodies or enzymes, may lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.
In view of the difficulties discussed above, there remains a need for methods of sterilizing preparations of one or more glycosidases that are effective for reducing the level of active biological contaminants or pathogens without an adverse effect on the preparation.
The above references are incorporated by reference herein where appropriate for appropriate teachings of additional or alternative details, features and/or technical background.
An object of the invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
Accordingly, it is an object of the present invention to provide methods of sterilizing preparations of glycosidases by reducing the level of active biological contaminants or pathogens without adversely affecting the preparation. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.
In accordance with these and other objects, a first embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising irradiating the preparation of one or more glycosidases with radiation for a time effective to sterilize the material at a rate effective to sterilize the material and to protect the material from radiation.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) adding to a preparation of one or more glycosidases at least one stabilizer in an amount effective to protect the preparation of one or more glycosidases from radiation; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the material.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of one or more glycosidases to a level effective to protect the preparation of one or more glycosidases from radiation; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) reducing the temperature of a preparation of one or more glycosidases to a level effective to protect the preparation of one or more glycosidases from radiation; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) applying to the preparation of one or more glycosidases a stabilizing process selected from the group consisting of: (a) reducing the residual solvent content of a preparation of one or more glycosidases, (b) adding to the preparation of one or more glycosidases at least one stabilizer, and (c) reducing the temperature of the preparation of one or more glycosidases; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases, wherein the stabilizing process and the rate of irradiation are together effective to protect the preparation of one or more glycosidases from radiation.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more glycosidases that is sensitive to radiation comprising: (i) applying to the preparation of one or more glycosidases at least two stabilizing processes selected from the group consisting of: (a) reducing the residual solvent content of a preparation of one or more glycosidases, (b) adding to the preparation of one or more glycosidases at least one stabilizer, and (c) reducing the temperature of the preparation of one or more glycosidases; and (ii) irradiating the preparation of one or more glycosidases with radiation at an effective rate for a time effective to sterilize the preparation of one or more glycosidases, wherein the stabilizing processes may be performed in any order and are together effective to protect the preparation of one or more glycosidases from radiation.
The invention also provides a biological composition comprising at least one preparation of one or more glycosidases and a least one stabilizer in an amount effective to preserve the preparation of one or more glycosidases for its intended use following sterilization with radiation.
The invention also provides a biological composition comprising at least one preparation of one or more glycosidases in which the residual solvent content has been reduced to a level effective to preserve the preparation of one or more glycosidases for its intended use following sterilization with radiation.
The invention also provides a biological composition comprising at least one preparation of one or more glycosidases and at least one stabilizer in which the residual solvent content has been reduced and wherein the amount of stabilizer and level of residual solvent content are together effective to preserve the preparation of one or more glycosidases for its intended use following sterilization with radiation.
The invention also provides a biological composition comprising at least one preparation of one or more glycosidases wherein the total protein concentration of the preparation is effective to preserve the preparation of one or more glycosidases for its intended use following sterilization with radiation.
The invention also provides a method of treating a disease, disorder or deficiency of glycosidase comprising the administration of at least one preparation of one or more glycosidases that has been sterilized by one or more of the methods described herein that is effective to preserve the preparation for its intended use following sterilization with radiation.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims.